Fig. S1. Accumulation of GPI−GFP in the trans-Golgi network (TGN) after cold temperature block at 20°C. A PC12 cell expressing GPI−GFP (green) were incubated for 30 hours at 20°C, fixed in 3.5% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS, and stained with anti-p115 and Alexa Fluor 567 secondary antibodies (red). Scale bar: 5 m.

Fig. S2. Three-dimensional analysis of the colocalization between SYN−GFP and CgA−RFP during post-Golgi transport. (A) The colocalization between SYN−GFP (green) and CgA−RFP (red) was analyzed through the z-axis in cells expressing both proteins. The images taken through the z-axis were combined to generate a three-dimensional (3D) image. As shown in the inset, SYN−GFP and CgA−RFP co-accumulate in various sizes of intermediate compartments 0 minutes after 20°C block. (B) The 3D image shows an increased merge between SYN−GFP and CgA−RFP at 10 minutes after release from 20°C block. More SYN−GFP appears to merge with CgA−RFP at 10 minutes (inset). (C) SYN−GFP and CgA−RFP begin to separate from each other 20 minutes after the release. 3D image shows the budding off of CgA−RFP from the SYN−GFP-containing compartment (inset). (D) At 30 minutes, most CgA−RFP is separated from SYN−GFP, showing little colocalization with SYN−GFP. The inset shows vesicles having only CgA−RFP. Insets on top left represent non-reconstructed dual channel fluorescent images. Combined 3D images are shown on bottom left. Scale bar: 5 m.

Fig. S3. The morphology of Golgi complex before and after 20°C block. (A) The morphology of Golgi complex stained with anti-p115 antibody was imaged at 37°C and 20°C. (B) Quantitative analysis of Golgi morphology at 37°C and 20°C.

Fig. S4. SYN−GFP, but not CgA−RFP, is present in the TfnR-positive recycling endosomes during 20°C block and release. (A) The transferrin receptor (TfnR, red) in SYN−GFP (green)-expressing cells was stained with anti-TfnR monoclonal antibody. (B) The TfnR-positive compartment (green) in cells expressing CgA−RFP (red) was visualized by immunostaining. The OCCs between SYN−GFP and TfnR (C) and between CgA−RFP and TfnR (D) were measured. The average OCC ± s.e.m. was calculated from three different experiments (n=30). Scale bar: 5 m.

Fig. S5. Steady state distribution of VAChT−GFP, SYN-G/RFP and CgA−RFP in PC12 cells. 3D images of colocalization (lower panels) between (A) VAChT−GFP (green) and CgA−RFP (red), (B) VAChT−GFP and SYN−RFP, or (C) SYN−GFP (green) and CgA−RFP (red) at steady state (Inset: magnified colocalization area). (D) The OCCs between VAChT−GFP, SYN-R/GFP and CgA−RFP were measured from PC12 cells transfected with those constructs and fixed 18−20 hours after transfection. The average OCC ± s.e.m. was calculated from three different experiments (n=30, *P<0.05). Scale bar: 5 m.

Fig. S6. Steady state distribution of VAChT−GFP, SYN-G/RFP and CgA−RFP with respect to the Golgi and the early endosomes. Sub-TGN compartments containing VAChT−GFP (green) (A) and SYN−GFP (green) (B) at steady state were compared with those stained with antibody against cis−medial Golgi (p115, red). (C) Steady state distribution of CGA−RFP (red) relative to p115 (green). Early endosomes were labeled with an antibody against TfnR (red) and compared with the steady state distribution of (D) VAChT−GFP (green) and (E) SYN−GFP (green). (F) CgA−RFP (red) was compared with TfnR (green) in the early endosome. (G) The OCCs between p115 and VAChT−GFP, SYN−GFP or CgA−RFP were measured from cells under normal condition. (H) The OCCs between TfnR and VAChT−GFP, SYN−GFP or CgA−RFP were measured from cells under normal condition. *P<0.05. Scale bar: 5 m.

Movie 1. Live cell movies of VAChT−GFP and CgA−RFP after 20°C block and release in undifferentiated PC12 cells. Undifferentiated PC12 cells were transfected with cDNAs encoding VAChT−GFP and CgA−RFP for 12 hr at 37°C and subsequently incubated for 30 hours at 20°C to arrest post-Golgi vesicle trafficking. Then, the cells were moved to a confocal microscope warmed to 37°C to record time-lapse movements of VAChT−GFP and CgA−RFP after release from the 20°C block. The image size is 22x22 m. The movie contains 1500 frames played at eight frames per second.